Use of cannabinoids in the treatment of epilepsy

ABSTRACT

The present invention relates to the use of a therapeutically effective amount of cannabidiolic acid (CBDA) in the treatment of epilepsy. In one embodiment the CBDA is used in the treatment of generalised seizures, preferably tonic-clonic seizures. Preferably the CBDA used is in the form of a botanical drug substance in which the CBDA content is greater than 60%, and most preferably, it is a highly purified extract of cannabis such that the CBDA is present at greater than 95%, through 96% and 97% to most preferably, greater than 98% of the total extract (w/w) and the other components of the extract are characterised. In particular the cannabinoids tetrahydrocannabinol (THC) or tetrahydrocannabinol acid (THCA) have been substantially removed. Alternatively, the CBDA may be synthetically produced.

This application is a National Stage application under 35 U.S.C. § 371of International Application No. PCT/GB2016/052340, having anInternational Filing Date of Jul. 29, 2016, which claims the benefit ofGB Serial No. 1514079.1 filed Aug. 10, 2015. This disclosure of theprior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

FIELD OF THE INVENTION

The present invention relates to the use of a therapeutically effectiveamount of cannabidiolic acid (CBDA) in the treatment of epilepsy. In oneembodiment the CBDA is used in the treatment of generalised seizures,preferably tonic-clonic seizures.

Preferably the CBDA used is in the form of a botanical drug substance inwhich the CBDA content is greater than 60%, and most preferably, it is ahighly purified extract of cannabis such that the CBDA is present atgreater than 95%, through 96% and 97% to most preferably, greater than98% of the total extract (w/w) and the other components of the extractare characterised. In particular the cannabinoids tetrahydrocannabinol(THC) or tetrahydrocannabinol acid (THCA) have been substantiallyremoved. Alternatively, the CBDA may be synthetically produced.

In use the CBDA may be used concomitantly with one or more otheranti-epileptic drugs (AED). Alternatively the CBDA may be formulated foradministration separately, sequentially or simultaneously with one ormore AED or the combination may be provided in a single dosage form.Where the CBDA is formulated for administration separately, sequentiallyor simultaneously it may be provided as a kit or together withinstructions to administer the one or more components in the mannerindicated. It may also be used as the sole medication, i.e. as amonotherapy.

BACKGROUND TO THE INVENTION

Epilepsy occurs in approximately 1% of the population worldwide,(Thurman et al., 2011) of which 70% are able to adequately control theirsymptoms with the available existing anti-epileptic drugs (AED).However, 30% of this patient group, (Eadie et al., 2012), are unable toobtain seizure freedom using the AED that are available and as such aretermed as suffering from intractable or “treatment-resistant epilepsy”(TRE).

Intractable or treatment-resistant epilepsy was defined in 2009 by theInternational League Against Epilepsy (ILAE) as “failure of adequatetrials of two tolerated and appropriately chosen and used AED schedules(whether as monotherapies or in combination) to achieve sustainedseizure freedom” (Kwan et al., 2009).

Individuals who develop epilepsy during the first few years of life areoften difficult to treat and as such are often termedtreatment-resistant. Children who undergo frequent seizures in childhoodare often left with neurological damage which can cause cognitive,behavioral and motor delays.

Childhood epilepsy is a relatively common neurological disorder inchildren and young adults with a prevalence of approximately 700 per100,000. This is twice the number of epileptic adults per population.

When a child or young adult presents with a seizure, investigations arenormally undertaken in order to investigate the cause. Childhoodepilepsy can be caused by many different syndromes and genetic mutationsand as such diagnosis for these children may take some time.

The main symptom of epilepsy is repeated seizures. In order to determinethe type of epilepsy or the epileptic syndrome that a patient issuffering from, an investigation into the type of seizures that thepatient is experiencing is undertaken. Clinical observations andelectroencephalography (EEG) tests are conducted and the type(s) ofseizures are classified according to the ILAE classification describedbelow and in FIG. 1.

The International classification of seizure types proposed by the ILAEwas adopted in 1981 and a revised proposal was published by the ILAE in2010 and has not yet superseded the 1981 classification. FIG. 1 isadapted from the 2010 proposal for revised terminology and includes theproposed changes to replace the terminology of “partial” with “focal”.In addition the term “simple partial seizure” has been replaced by theterm “focal seizure where awareness/responsiveness are not impaired” andthe term “complex partial seizure” has been replaced by the term “focalseizure where awareness/consciousness are impaired”.

From FIG. 1 it can be seen that Generalised seizures, where the seizurearises within and rapidly engages bilaterally distributed networks, canbe split into six subtypes: Tonic-Clonic (grand mal) seizures; Absence(petit mal) Seizures; Clonic Seizures; Tonic Seizures; Atonic Seizuresand Myoclonic Seizures.

Focal (partial) seizures where the seizure originates within networkslimited to only one hemisphere, are also split into sub-categories. Herethe seizure is characterized according to one or more features of theseizure, including aura, motor, autonomic and awareness/responsiveness.Where a seizure begins as a localized seizure and rapidly evolves to bedistributed within bilateral networks this seizure is known as aBilateral convulsive seizure, which is the proposed terminology toreplace Secondary Generalized Seizures (generalized seizures that haveevolved from focal seizures and no longer remain localized).

Focal seizures where the subject's awareness/responsiveness is alteredare referred to as focal seizures with impairment and focal seizureswhere the awareness or responsiveness of the subject is not impaired arereferred to as focal seizures without impairment.

Focal seizures may occur in epilepsy syndromes including: Lennox-GastautSyndrome; Tuberous Sclerosis Complex; Dravet Syndrome; CDKL5; Neuronalceroid lipofuscinoses (NCL); febrile infection related epilepsy syndrome(FIRES); Aicardi syndrome and brain abnormalities.

Epileptic syndromes often present with many different types of seizureand identifying the types of seizure that a patient is suffering from isimportant as many of the standard AED are targeted to treat or are onlyeffective against a given seizure type/sub-type.

Common AED defined by their mechanisms of action are described in thefollowing tables:

TABLE 1 Examples of narrow spectrum AED Narrow- spectrum AED MechanismIndication Phenytoin Sodium channel Complex partial Tonic-clonicPhenobarbital GABA/Calcium channel Partial seizures Tonic-clonicCarbamazepine Sodium channel Partial seizures Tonic-clonic Mixedseizures Oxcarbazepine Sodium channel Partial seizures Tonic-clonicMixed seizures Gabapentin Calcium channel Partial seizures Mixedseizures Pregabalin Calcium channel Adjunct therapy for partial seizureswith or without secondary generalisation Lacosamide Sodium channelAdjunct therapy for partial seizures Vigabatrin GABA Secondarilygeneralized tonic- clonic seizures Partial seizures Infantile spasms dueto West syndrome

TABLE 2 Examples of broad spectrum AED Broad- spectrum AED MechanismIndication Valproic acid GABA/Sodium First-line treatment for tonic-channel clonic seizures, absence seizures and myoclonic seizuresSecond-line treatment for partial seizures and infantile spasms.Intravenous use in status epilepticus Lamotrigine Sodium channel Partialseizures Tonic-clonic Seizures associated with Lennox-Gastaut syndromeEthosuximide Calcium channel Absence seizures Topiramate GABA/SodiumSeizures associated with channel Lennox-Gastaut syndrome ZonisamideGABA/Calcium/ Adjunctive therapy in adults with Sodium channelpartial-onset seizures Infantile spasm Mixed seizure Lennox-Gastautsyndrome Myoclonic Generalised tonic-clonic seizure LevetiracetamCalcium channel Partial seizures Adjunctive therapy for partial,myoclonic and tonic-clonic seizures Clonazepam GABA Typical and atypicalabsences Infantile myoclonic Myoclonic seizures Akinetic seizuresRufinamide Sodium channel Adjunctive treatment of partial seizuresassociated with Lennox-Gastaut syndrome

TABLE 3 Examples of AED used specifically in childhood epilepsy AEDMechanism Indication Clobazam GABA Adjunctive therapy in complex partialseizures Status epilepticus Myoclonic Myoclonic-absent Simple partialComplex partial Absence seizures Lennox-Gastaut syndrome StiripentolGABA Severe myoclonic epilepsy in infancy (Dravet syndrome)

Over the past forty years there have been a number of animal studies onthe use of the non-psychoactive cannabinoid cannabidiol (CBD) to treatseizures. For example, Consroe et al., (1982) determined that CBD wasable to prevent seizures in mice after administration of pro-convulsantdrugs or an electric current.

Studies in epileptic adults have also occurred in the past forty yearswith CBD. Cunha et al. reported that administration of CBD to eightadult patients with generalized epilepsy resulted in a marked reductionof seizures in 4 of the patients (Cunha et al., 1980).

A study in 1978 provided 200 mg/day of pure CBD to four adult patients,two of the four patients became seizure free, whereas in the remainderseizure frequency was unchanged (Mechoulam and Carlini, 1978).

In contrast to the studies described above, an open label study reportedthat 200 mg/day of pure CBD was ineffective in controlling seizures intwelve institutionalized adult patients (Ames and Cridland, 1986).

Based on the fact that chronologically the last study to look at theeffectiveness of CBD in patients with epilepsy suggested that CBD wasunable to control seizures, there may be less of an expectation that CBDmight be useful as an anti-convulsant agent.

In the past forty years of research there have been over thirty drugsapproved for the treatment of epilepsy none of which are cannabinoids.Indeed, there appears to have been a prejudice against cannabinoids,possibly due to the scheduled nature of these compounds and/or the factthat THC, which is a known psychoactive, has been ascribed as apro-convulsant (Consroe et al., 1977).

A paper published recently suggested that cannabidiol-enriched cannabismay be efficacious in the treatment of epilepsy. Porter and Jacobson(2013) report on a parent survey conducted via a Facebook group whichexplored the use of cannabis which was enriched with CBD in childrenwith treatment-resistant epilepsy. It was found that sixteen of the 19parents surveyed reported an improvement in their child's epilepsy. Thechildren surveyed for this paper were all taking cannabis extracts thatwere purported to contain CBD in a high concentration although theamount of CBD present and the other constituents including THC andnon-cannabinoid components such as terpenes were not known for many ofthe cases. Indeed, whilst CBD levels ranged from 0.5 to 28.6 mg/kg/day(in those extracts tested), THC levels as high as 0.8 mg/kg/day werereported.

Providing children with TRE with a cannabis extract that comprises THC,which has been described as a pro-convulsant (Consroe et al., 1977), ata potentially psychoactive dose of 0.8 mg/kg/day is not desirable.

Whilst decoctions of cannabis which will contain CBDA as well as THCAalong with other cannabinoids and non-cannabinoid components have beenused in epilepsy, treatments have not focussed on isolated or highlypurified CBDA. Rather the recent focus has been on the use of thedecarboxylated form of CBDA, CBD in the treatment of epilepsy.

CBDA has however been found to be effective in the treatment of nauseaas is shown in WO 2003/063847 and as a TNF alpha inhibitor suggested foruse in treating immunomodulatory and anti-inflammatory conditions as isshown in WO 2002/064109.

The patent application GB 2,495,118 describes the use of a compositioncomprising CBDV and CBD for use in the treatment of epilepsy.Furthermore the application WO 2011/121351 describes the use of CBDV inthe treatment of epilepsy. Both documents describe the use of a CBDVbotanical drug substance which comprises a small quantity ofundecarboxylated CBD as CBDA. The CBDA is present in very small amountsand as such is not present in therapeutically effective amounts.

The patent application US 2015/126595 describes the use of a transdermalcomposition comprising cannabinoids including CBDA.

Patent applications CA 2,859,934 and CA 2,737,447 both describe amedicinal cannabis chemovar which comprises the compound CBDA. It isreadily understood that all cannabis plants produce cannabinoids intheir acid form which are then readily decarboxylated to produce thetraditionally recognised active form CBD.

Whilst CBD now appears to be a promising candidate as an anti-epilepticdrug there are a number of potential limitations including: the relativelarge doses that appear necessary; and CBD's relatively poorbioavailability.

Therefore it is desirable to find other compounds which may demonstrateactivity and/or specificity to particular seizure sub-types and whichmight be administered in lower concentrations. This has the benefit ofsmaller administration forms and with improved bioavailability lowerdose may be required and onset to action may be quicker.

BRIEF SUMMARY OF THE DISCLOSURE

In accordance with a first aspect of the present invention there isprovided a therapeutically effective amount of cannabidiolic acid (CBDA)for use in the treatment of epilepsy.

In one embodiment the epilepsy is generalised epilepsy. More preferablythe epilepsy is characterized by tonic-clonic seizures.

A therapeutically effective amount is preferably at least 0.1 mg,preferably at least 0.5 mg, more preferably at least 1 mg, morepreferably still at least 20 mg or more.

The CBDA used may be in the form of a botanical drug substance in whichthe CBDA content is greater than 60%, and most preferably, it is ahighly purified extract of cannabis such that the CBDA is present atgreater than 95%, through 96% and 97% to most preferably, greater than98% of the total extract (w/w) and the other components of the extractare characterised. In particular the cannabinoids tetrahydrocannabinol(THC) or tetrahydrocannabinol acid (THCA) have been substantiallyremoved. Preferably the highly purified extract comprises less than 1%(w/w) tetrahydrocannabinol (THC) or tetrahydrocannabinol acid (THCA).

Alternatively, the CBDA may be synthetically produced.

The CBDA may also be used concomitantly with one or more othercannabinoids. Preferably the CBDA is used with CBD.

Where CBDA is used in combination with CBD ratios of between 9:1 to 1:9(CBDA:CBD) are preferred. Ranges of ratios include 8:2 to 2:8(CBDA:CBD); 7:3 to 3:7 (CBDA:CBD); 6:4 to 4:6 (CBDA:CBD); and 1:1(CBDA:CBD) and any ranges there between.

In a further embodiment of the invention the CBDA is used concomitantlywith one or more other anti-epileptic drugs (AED).

The CBDA may be used at a daily dose of less than 1000 mg. Preferably,the daily dose of CBDA is less than 800 mg, preferably less than 600 mg,and more preferably less than 400 mg.

The daily dose may be less than 200 mg, less than 100 mg and as littleas 10 mg or 1 mg may be used.

As the cannabinoid CBDA is more bioavailable than its neutral form CBD,it is likely that a far lower dose of CBDA will be required incomparison with CBD when treating the same indication. For exampleproviding a human with a dose of 20 mg/kg of CBD to treat epilepsy maybe effective, whereas the dose of CBDA required may be a log fold lower.

Clearly such lower doses have benefits in treatment.

Furthermore the greater bioavailability of CBDA may mean that it can actmore quickly than CBD. In other words the cannabinoid CBDA may have alower T_(max) than CBD. This quality could lead to useful combinationproducts which comprise CBDA in combination with CBD. The CBDA may beuseful in providing a rapid onset effect whereas the CBD may be usefulin providing a sustained effect.

Ratioed amounts of CBDA to CBD, where the CBDA is the predominantcannabinoid are envisaged these include ranges from 95:5 to 55:45(CBDA:CBD).

Alternatively the CBDA and CBD may be present in substantially equalamount namely 55:45 to 45:55 (CBDA:CBD). In yet a further embodiment theCBD may be the predominant cannabinoid and the range may be from 45:55to 20:80 (CBDA:CBD).

Furthermore the faster acting CBDA may be a useful candidate for use inthe treatment of epilepsy which requires immediate emergency treatmentsuch as acute seizures or status epilepticus. Preferably the CBDA isadministered via the parenteral route, for example by injection into thevein or the muscle.

In accordance with a second aspect of the present invention there isprovided a method of treating epilepsy comprising administering atherapeutically effective amount of cannabidiolic acid (CBDA) to asubject.

Preferably the subject is a human.

In accordance with a third aspect of the present invention there isprovided a composition for use in the treatment of epilepsy comprising atherapeutically effective amount of cannabidiolic acid (CBDA), and oneor more pharmaceutically acceptable excipients.

It is envisaged that the composition be administered as one or more of:an oral liquid solution, solid, semi-solid, gel, injection, spray,aerosol, inhaler, vaporiser, enema or suppository. Such medicamentscould be administered via the oral, buccal, sublingual, parenteral,respiratory, nasal and distal rectum route.

DEFINITIONS

Definitions of some of the terms used to describe the invention aredetailed below:

The cannabinoids described in the present application are listed belowalong with their standard abbreviations.

TABLE 4 Cannabinoids and their abbreviations CBD Cannabidiol

CBDA Cannabidiolic acid

THC Tetrahydrocannabinol

THCA Tetrahydrocannabinolic acid

The table above is not exhaustive and merely details the cannabinoidswhich are identified in the present application for reference. So farover 60 different cannabinoids have been identified and thesecannabinoids can be split into different groups as follows:Phytocannabinoids; Endocannabinoids and Synthetic cannabinoids (whichmay be novel cannabinoids or synthetically produced phytocannabinoids orendocannabinoids).

Patent application number WO 2004/026857 describes the analysis ofhighly purified CBDA. The CBDA is described as being purified to begreater than 98% pure, with less than 0.1% CBD, 0.3% THCA, and less than0.1% THC.

“Phytocannabinoids” are cannabinoids that originate from nature and canbe found in the cannabis plant. The phytocannabinoids can be isolatedfrom plants to produce a highly purified extract or can be reproducedsynthetically.

“Highly purified cannabinoid extracts” are defined as cannabinoids thathave been extracted from the cannabis plant and purified to the extentthat other cannabinoids and non-cannabinoid components that areco-extracted with the cannabinoids have been substantially removed, suchthat the highly purified cannabinoid is greater than or equal to 98%(w/w) pure.

“Synthetic cannabinoids” are compounds that have a cannabinoid orcannabinoid-like structure and are manufactured using chemical meansrather than by the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which

FIG. 1 shows the ILAE Proposal for Revised Terminology for Organisationof Seizures and Epilepsies 2010;

FIGS. 2 A, B, C and D show the effect of cannabinoids on PTZ-inducedgeneralised seizures;

FIG. 3 shows the effect of CBDA on D. discoideum cell growth;

FIG. 4 shows the effect of CBD on D. discoideum cell growth;

FIG. 5 shows secondary plots for the effect of CBDA and CBD on D.discoideum growth; and

FIG. 6 shows the HPLC trace of the CBDA extract of Example 3.

LEGENDS TO THE FIGURES

FIG. 2: Panels A-D illustrate the effect of CBDA (10-100 mg/kg), CBD(100 mg/kg), and CBD+CBDA (9:1 ratio) on seizure severity (A),percentage mortality (B), percentage of animals exhibiting tonic-clonicseizures (C) and latency to seizure onset (D). In panel A, medianseizure severity is represented by a thick grey horizontal line, 25thand 75th percentiles by the black box and whiskers indicate the minimumand maximum values. In panel D, onset latency is presented as medianwith IQR. Statistical testing was performed using either aKruskal-Wallis with post-hoc Mann-Whitney U-tests (panel A and D) orChi-squared with post-hoc Fisher exact tests (panel B and C): P≤0.1 (#);P≤0.05 (*); P≤0.01 (**); P≤0.001 (***); n=15 per group.

FIG. 3: Growth was measured over a seven day period in the presence ofCBDA at concentrations ranging from 0.02 μM to 20 μM. A secondary plotof cell density at 168 hours was used to calculate an IC50 of 0.30 μM.

FIG. 4: Growth was measured over a seven day period in the presence ofCBD (from GW Pharmaceuticals) at concentrations ranging from 0.25 μM to20 μM. A secondary plot of cell density at 144 hours was used tocalculate an IC50 of 1.63 μM.

FIG. 5: Cannabinoids have a potency order of CBDA>CBD.

FIG. 6: The CBDA botanical drub substance shown in the HPL tracecomprised a CBDA content of 62.4% w/w and other cannabinoids measuredincluded CBD—6.9% (w/w), THC—0.7% (w/w) and cannabichromene (CBC)—0.5%(w/w).

DETAILED DESCRIPTION Example 1: An In Vivo Evaluation of CBDA in theAcute Pentylenetetrazole (Ptz) Model of Generalised Seizure

Materials and Methods

Animals

Adult male Wistar Kyoto rats were used in the acute PTZ model of seizure(>P24, 70-110 g). Animals were housed five per cage in a heat regulatedroom (21° C.) on a 12:12 h day/night cycle (lights on 0800) in 50%humidity and given ad libitum access to standard laboratory chow (PCDMod C, Special Diet Services, Wiltham, UK) and water. All procedureswere undertaken during white light hours.

Pharmaceutical Formulation

A PTZ (Sigma-Aldrich, Poole, UK) stock solution was made in 0.9% w/vNaCl for the experimental procedure. CBD (batch number, CBD-CG-1001; GWPharmaceuticals, Salisbury, UK) and CBDA (batch number, CBDA040912; GWPharmaceuticals) stocks were made in a 2:1:17 vehicle of ethanol,cremophor and saline.

Formulation analysis was undertaken to determine whether CBDAdecarboxylated to CBD because of temperature and/or the excipients inthe formulation. Analysis revealed CBDA was not converted to CBD duringformulation. Therefore, CBDA was not modified during formulation, and a2:1:17 ratio can be used in future investigations for thisphytocannabinoid.

PTZ-Induced Model of Generalised Seizure

PTZ antagonises GABA inhibition via binding to thet-butyl-bicycl-phosphorothionate site of GABAA receptors. Moreover, thischemically-induced model can be indicative of effects against absenceseizures. PTZ (90 mg/kg) was used to induce seizures in adult maleWistar rats (n=15 per group) with experiment dose randomised using aLatin square design. Animals were placed in their 6 L Perspex tanks andallowed to acclimatise to their environment for 10 min, before receivingone of the cannabinoid doses (see Table 5) in vehicle, or volume-matcheddose of vehicle alone to serve as a negative control. 60 min after testcompound or vehicle administration, animals were injected with PTZ (90mg/kg, i.p.) to induce seizures and animal behaviour was recorded for 30min.

TABLE 5 Doses of cannabinoid Dose (mg/kg) CBDA CBD Vehicle — — CBDA 10 —CBDA 50 — CBDA 100  — CBD — 100 CBDA/CBD 10  90

Videos of PTZ-induced seizures were scored offline with a standardseizure severity scale appropriate for generalised seizures (Table 6).

The human dose equivalent (HED) can be estimated using the followingformula:

${HED} = {{Animal}\mspace{14mu}{dose}\mspace{14mu}\left( {{mg}\text{/}{kg}} \right)\mspace{14mu}{multiplied}\mspace{14mu}{by}\mspace{14mu}\frac{{Animal}\mspace{14mu} K_{m}}{{Human}\mspace{14mu} K_{m}}}$

The K_(m) for a rat is 6 and the K_(m) for a human is 37.

The K_(m) for a dog (Example 3) is 20.

Thus a 10 mg/Kg dose in a rat would equate to a human dose of about 1.6mg/kg. A 50 mg/kg dose in a rat would equate to a human dose of about8.1 mg/kg. A 100 mg/kg dose in a rat would equate to a human dose ofabout 16.2 mg/kg.

TABLE 6 Seizure severity scoring scale. Seizure Righting scoreBehavioural expression reflex 0 No changes to behaviour Preserved 0.5Abnormal behaviour (sniffing, excessive washing, Preserved orientation)1 Isolated myoclonic jerks Preserved 2 Atypical clonic seizure Preserved3 Fully developed bilateral forelimb clonus Preserved 3.5 Forelimbclonus with tonic component and body Preserved twist 4 Tonic-clonicseizure with suppressed tonic phase Lost 5 Fully developed tonic-clonicseizure LostData Analysis

Videos of seizure behaviour generated from the custom builtobservational system were scored offline according to seizure scalesappropriate for PTZ model (Table 6) using Observer Video-Pro software(Noldus, Wageningen, The Netherlands). For the PTZ model of seizure,intra- and inter-observer agreements of behaviour scoring were assessedusing the reliability analysis function of the observer Video-Prosoftware: 1 s tolerance window; Cohen's Kappa coefficient ≥0.95.

Specific markers of seizure behaviour and development were assessed andcompared between vehicle control and drug groups. The latency (s) toseizure onset and the percentage of animals that developed tonic-clonicseizures was noted (see Table 6). In addition, the maximum seizureseverity and the percentage mortality in each group were determined forthe acute PTZ model of generalised seizure.

Statistical Analysis

The effect of drug on latency to seizure onset and maximum seizureseverity were assessed using Krustal-Wallis with post-hoc Mann-WhitneyU-tests. Drug effects on the percentage of animals that developedtonic-clonic seizures and percentage mortality were assessed usingChi-squared with post-hoc Fisher exact tests. In all cases, P≤0.05 wasconsidered significant.

Results

The objective of the present Example was to examine the anti-convulsantpotential of CBDA in the PTZ-induced acute model of generalised seizure,with a comparator CBD dose. Additionally, CBD and CBDA in a ratio of 9:1was included to investigate possible interactions between CBD and CBDA.

Cannabinoid treatment significantly reduced seizure severity in theacute PTZ-induced model of generalised seizure (FIG. 2. Panel A;H=14.31, P≤0.05), where 100 mg/kg CBDA (P≤0.05) and a CBD/CBDA ratio(P≤0.05) exhibited significant anti-convulsant effects vs vehiclecontrol.

Mortality was significantly reduced (FIG. 2. Panel B; X2(6)=30.51,P≤0.0001) following administration of 100 mg/kg CBD (P≤0.01) and a trendtowards a significant reduction for 100 mg/kg CBDA (P=0.0656) and aCBD/CBDA ratio (P=0.0656).

Cannabinoid treatment also significantly reduced incidence oftonic-clonic seizures (FIG. 2. Panel C; X2(6)=17.178, P≤0.01) whereadministration of 100 mg/kg of CBDA resulted in a trend towards areduction in tonic-clonic seizures (P≤0.1).

Finally, analysis revealed cannabinoid administration significantaffected latency to seizure onset in the PTZ-induced model (FIG. 2.Panel D; H=37.37, P≤0.0001), with 100 mg/kg CBD (P≤0.05), CBD/CBDA (9:1ratio; P≤0.05) significantly increasing latency to onset. Whereas CBDA(100 mg/kg) showed a trend towards increasing the latency to seizureonset (P=0.0929).

Conclusions

As predicted by previous studies, CBD exerted anti-convulsant effects inthis model of acute generalised seizure and so demonstrates thecontinued validity of the model to reveal anti-convulsant effects ofplant cannabinoids.

Notably, in three of the four parameters measured CBDA producedsignificant anti-convulsant effects and were statistically moreeffective than CBD at an equivalent dose.

For example, Panel A of FIG. 2 describes the effect of CBDA and CBD onthe seizure severity. The median score for the maximum seizure severitythat the animals experienced with 100 mg/kg CBDA was 3 (Table 6—fullydeveloped bilateral forelimb clonus, with righting reflex preserved) andthe median for 100 mg/kg CBD was 5 (Table 6—fully developed tonic-clonicseizure, with righting reflex lost). This shows that CBDA was able toprevent the animals from suffering from more severe types of seizurethan CBD was.

Panel C additionally demonstrates that CBDA at 100 mg/kg was able toprevent tonic-clonic seizures from developing in more animals comparedto CBD at 100 mg/kg. Indeed the data for 100 mg/kg CBDA was the onlystatistically significant data in this parameter. This suggests thatCBDA will be more effective than CBD at preventing or treating epilepsy,particularly tonic-clonic seizures from developing.

Co-administration of CBDA with CBD, in an exemplary 9:1 ratio,demonstrated that the combination was also effective as ananti-convulsant. Since the plant naturally produces CBDA and this can bedecarboxylated, this opens up the possibility of using partiallydecarboxylated phytocannabinoids or extracts thereof, in given ratios.Such ratios may be beneficial for a number of reasons. These includetargeting different types of seizures e.g. CBD for partial seizures andCBDA for generalised seizures may be beneficial based on their differentactivities in animal models of epilepsy. Also, the difference in thelipophilicity or bioavailability of the two compounds may enablecombinations to be developed with different release profiles e.g. CBDAmay be quicker acting than CBD and more bioavailable than CBD (seeExample 3).

This Example demonstrates for the first time that the isolated or highlypurified cannabinoid CBDA has anti-convulsant effects and as suchfurther investigation in other models of seizure and epilepsy arewarranted in order to determine the full extent of its efficacy.

In order to consider whether CBD and CBDA act by similar mechanisms andhave similar potency the applicant conducted a study on a new model ofDictyostelium discoideum.

Example 2: Use of Dictyostelium Discoideum Model to Identify MolecularTargets of Cannabinoids and their Use in Epilepsy

Introduction

Dictyostelium discoideum is an amoeba, listed by the US NationalInstitute of Health as a biomedical model system (Williams et al. 2006).It has a cellular structure typical of eukaryotes, with nuclei, Golgi,mitochondria and endoplasmic reticulum and its haploid genome has beenfully characterised and annotated (Dictybase.org) including descriptionsof each protein, the phenotype of mutants lacking each protein andrelated published material. D. discoideum can be grown in liquid cultureas single cells or allowed to progress into multi-cellular developmentupon starvation with the formation of a multi-cellular fruiting body.

D. discoideum has been developed to better understand the molecularmechanisms by which diverse drugs and chemicals exert their effects, toidentify more potent or safer compounds, and to characterise thecellular role of human proteins

This range of methodologies has enabled D. discoideum to be used as avaluable model in diverse areas in molecular pharmacology andpharmacogenetics. In these research areas, the primary target of eitherestablished or new pharmaceutical compounds is often unclear, andcompounds often have off-target side effects that remainuncharacterised, and which may result in costly late-stage drugattrition and potentially affecting patient compliance.

In epilepsy research, D. discoideum has been used to identify moleculareffects of valproic acid (Cunliffe et al 2015; Chang et al. 2012) andtranslated in vitro and in vivo mammalian models to demonstraterelevance to human health (Chang et al 2012, 2013, 2014). It is clearthat D. discoideum can be used to identify clinically relevanttherapeutic compounds for the treatment of epilepsy.

The present Example demonstrates the use of D. discoideum to identifythe molecular mechanism(s) of action of two cannabinoids, (CBD andCBDA), with relevance to seizure control.

Materials and Methods

Growth Assays

Wild type (Ax2) D. discoideum cells were grown in shaking culture (inHL5 medium) for two days prior to growth assays. Cells (9900 in 495 μlof media) were added to each well of a 24 well plate and 5 μl ofcannabinoid in DMSO (or DMSO only) was added to each well to achieveeach described concentration (1% final DMSO concentration), and cellswere maintained at 22° C. Cells were counted at 72 hours, and then every24 hours. Quadruplicate repeats were used for each concentration.

Development Assays

Wild type (Ax2) D. discoideum cells were grown in HL5 shaking culturefor two days prior to development assay. Cells were washed in phosphatebuffer (KK2; 20 mM Potassium phosphate buffer, pH 6.1), and 1×107 cellswere spread onto nitrocellulose filters (Millipore, Cork). Absorbentpads (Millipore, Cork), divided into quarters, were placed in 2 mlculture dishes and soaked with 0.5 ml KK2 containing the cannabinoids at20 μM. 1 mM Valproic acid was used as a positive control while KK2containing 1% DMSO was used as a solvent only control. Nitrocellulosefilters containing cells were quartered and place upon absorbent padsand maintained in a humid environment at 22° C. for 24 h. Fruiting bodymorphology was recorded using a dissection microscope and camera.

Bioinformatic Analysis

The amino acid sequence for potential H. sapien protein targets of thecannabinoids listed were obtained from Uniprot (www.uniprot.org).Homology searches of the D. discoideum genome were carried out using theonline Basic local alignment search (BLAST) algorithm available atdictybase.org. TMHMM server V. 2.0 transmembrane region predictorsoftware was used to determine possible transmembrane regions within theD. discoideum orthologue proteins. Regions of the proteins containinghighly conserved residues required for protein function were analysed bymultiple sequence alignment using ClustalW2.

Bacterial Plate Screen

SM agar plates were made with the addition of CBDA or CBD to finalconcentrations of 12.3 μM and 16.7 μM respectively. Heat killed (75° C.for 30 minutes) R. planticola was spread onto the plates and ˜50wild-type AX2 cells were added and left to grow at 22° C. Plates werechecked regularly for colonies.

Mutant REMI Library Screen

REMI library cells were grown in shaking culture (in HL5 medium) for twodays prior to screening. Cells (25,000 in 2 ml of media) were added toeach well of a 6 well plate and allowed to adhere for 20 minutes. Themedia from each well was replaced with media containing either: 4.88 μMCBDA or 9.47 μM CBD. Cells were screened in triplicate over a three weekperiod, maintained at 22° C. with the media being replaced every twodays. Potential resistant mutant colonies were isolated and transferredto bacterial plates. Isogenic cell lines were established fromindividual colonies on the bacterial plates.

Confirmation of Individual Mutant Resistance:

Clonal cells isolated from the library screen were grown in liquid media(HL5 medium) to produce a confluent 10 cm plate. Cells (10,000 in 495 μlof media) were added to each well of a 24 well plate and 5 μl ofcannabinoid in DMSO was added to each well to achieve either 4.88 μMCBDA or 9.47 μM CBD (1% final DMSO concentration), cells were maintainedat 22° C. Cells were monitored over a one week for their sensitivity tothe two cannabinoids.

Results

Growth Assays

It first needed to be determined if D. discoideum growth was sensitiveto the cannabinoids: cannabidiolic acid (CBDA) and cannabidiol (CBD). Inthese experiments, D. discoideum were exposed to a range ofconcentrations of each cannabinoid during growth in still culture over aone week period. All two cannabinoids inhibited D. discoideum cellgrowth in a dose dependent manner (FIGS. 3 to 5).

The growth inhibitory constant (IC50) for CBDA was 0.30 μM (FIG. 3),with 0.08 μM significantly inhibiting cell growth (P<0.05) and 20 μMblocking growth.

The growth inhibitory constant (IC50) for CBD was 1.63 uM (FIG. 4), with0.5 μM significantly inhibiting cell growth (P<0.05) and 20 μM blockinggrowth.

Comparison of all two cannabinoids IC50 values suggests CBDA is the mostpotent, with CBD showing an 8.7-fold reduction in potency. Thus theorder of potency for cannabinoids on D. discoideum cell growth isCBDA>CBD (FIG. 5).

Development Assays

The effects that CBDA and CBD had upon D. discoideum development wereinvestigated. This was achieved by placing cells in a nutrient depletedenvironment in the presence of CBDA or CBD at concentrations that blockcell growth (20 μM).

Cell development on a nitrocellulose filter over a 24 hour period in theabsence of cannabinoids gave rise to fruiting bodies consisting of sporeheads held above substrata by stalks. This developmental morphology isknown to be blocked by the widely used anti-epileptic, valproic acid (1mM), where cells were able to aggregate but unable to undergodevelopment to form fruiting bodies.

In contrast, D. discoideum cells treated with CBDA or CBD (20 μM) wereable to aggregate and develop to form mature fruiting bodies.

Bioinformatic Analysis

Known targets of CBDA and CBD in H. sapiens were then sought in order toidentify potential orthologues within the D. discoideum genome. Fromcurrent literature, 21 possible mammalian targets of CBDA and CBD havebeen published. Using human protein sequences corresponding eachpotential target, in combination with BLAST analysis, the D. discoideumgenome was searched for orthologous targets. Using this approach, 10possible D. discoideum orthologues were identified. Based uponsimilarity of protein sequence and size, and conservation of catalyticsites and motifs, three proteins have been identified for further study:

1: Equilibrative Nucleoside Transporter 1 (ENT1). This protein is apotential target for CBD and plays a role in adenosine transport. D.discoideum has three possible ENT1 orthologues, and all three have aputative multiple transmembrane structure found in the H. sapiensprotein. The three D. discoideum orthologues are 522, 482 and 430 aa insize, similar to the 456 aa H. sapiens ENT1 protein, and contain ahighly conserved motif located within first transmembrane region. Thismotif is found within this protein from many other species.

2: Monoacylglyceride lipase alpha (MAGLa). This protein is involved inthe endocannabinoid system. D. discoideum has one possible MAGLaorthologue. This orthologue is 409 aa, of similar size to the 303 aa H.sapien MAGLA protein. Both the D. discoideum and H. sapiens proteinshave a conserved catalytic serine, aspartate and histidine residue thatare important in enzymatic function that are widely conserved in manyother species.

3: Diacylglycerol lipase alpha (DAGLa). This protein is involved in theendocannabinoid system. D. discoideum has three possible orthologues.The three D. discoideum orthologues are 938, 856 and 826 aa in size,slightly smaller than the 1042 aa H. sapiens DAGLa protein. All three D.discoideum orthologues have the same conserved serine and aspartateresidues that are important in catalytic function, and these are widelyconserved in many other species.

Bacterial Plate Screen

It was determined if D. discoideum growth upon R. planticola bacterialplates was a viable method in which resistant REMI mutant library cellscould be isolated. Wild-type AX2 cells were grown upon heat killed R.planticola SM agar plates. Each SM agar plate contained CBDA or CBD at afinal concentration of 12.3 μM and 16.7 μM respectively. Followingincubation for 4 days, plates were assessed for cell survival (colonygrowth). No difference in colony number was found for every cannabinoidcompared to control (solvent only).

Mutant REMI Library Screen

Mutants were then identified within the library that showed resistanceto the cannabinoids during growth in liquid culture. The library cellswere grown over a three week period in the presence of 4.88 μM CBDA or9.47 μM CBD. After a two week period colonies of partially resistantcells were visible in library-derived plates. Partially resistant cellswere transferred to bacterial plates and passaged to ensure each mutantwas isogenic.

Confirmation of Individual Mutant Resistance

The resistance of each cell line was confirmed. All cell lines weretreated with either: CBDA or CBD at a final concentration of 4.88 μM and9.47 μM respectively and assessed after one week. Isogenic cell linesshowed some overlap of resistance to the different cannabinoids. Mutantcells were shown to have 3 basic phenotypes to each cannabinoid,classified as showing no resistance, weak resistance or partialresistance. Mutant cells were also found to have either resistance toone cannabinoid or to multiple cannabinoids.

Conclusions

The development of cannabinoids as novel therapeutic treatments forepilepsy provides an exciting new field of research, with real potentialfor improving health. A comprehensive understanding of the mechanisms ofaction and relative potency of these compounds are essential fortherapeutic development, to understand both how the compounds blockseizures and potential side effects. Traditional approaches to identifythese mechanisms are very complex and slow. As an alternative approach,D. discoideum has been used to identify mechanism of a widely usedtreatment, valproic acid, which has been verified in mammalian in vivomodels.

In this current study, it has been demonstrated that two cannabinoids,CBDA, and CBD block D. discoideum growth. Concentrations that affectgrowth are in the low μM range and are equivalent to the concentrationsshown to be anti-convulsant in animal models of seizure. This suggeststhat targets for all two cannabinoids are present in the D. discoideumgenome. This also suggests that the D. discoideum targets have a similarsensitivity to the cannabinoids that shown in mammalian models.

The growth inhibitory effect can then be employed in an unbiased screento identify these cannabinoid targets. Using a library of insertionalmutants, a pool of mutants can be grown in the presence of eachcannabinoid over a 21 day period. Mutants with insertions into genesencoding cannabinoid targets are likely to show resistance to thisgrowth inhibition and thus out-compete sensitive cells during thescreen. Identification of insertionally-inactivated genes in cannabinoidresistant colonies will identify molecular targets (and mechanism) ofthese cannabinoids in an unbiased approach. This screening approach inD. discoideum has been used to identify targets and mechanisms of arange of compounds.

D. discoideum is also widely used as a development model, where theformation of a fruiting body involves cell aggregation anddifferentiation. Pharmacological studies have used this developmentalprocess to identify drug mechanisms. In relation to the cannabinoidsstudied here, all two compounds had no effect on D. discoideumdevelopment, at concentrations shown to block growth. This firstlysuggests that the block in D. discoideum growth is not toxic, sincecells can develop, and thus that cannabinoid targets are likely to beinvolved in blocking cell growth or division (cytokinesis). This alsosuggests that D. discoideum development cannot be used to further studythese compounds. In combination with an unbiased approach to identifyingcannabinoid targets, D. discoideum also provides a useful model toinvestigate known mammalian targets.

It was found that a total of 25 mutant cell lines showed resistance togrowth inhibition. The range of resistant phenotypes to differentcannabinoids suggests that there are multiple genes involved

Example 3 Comparison of PK Data for CBD and CBDA from ToxicologicalStudies in Dogs

The objective of the studies was to determine the toxicity of CBD (inthe form of a substantially pure compound—greater than 95% purity) andCBDA (in the form of a botanical drug substance—greater than 60% CBDAw/w of the total extract and greater than 85% w/w of the totalcannabinoid content) following daily oral (gavage) administration to thedog.

This study was designed to meet the known requirements of EuropeanDirective 2001/83/EC and all subsequent amendments together with anyrelevant International Conference on Harmonisation (ICH) guidelines.

Blood samples for toxicokinetics (0.5 mL nominal) were taken from allanimals on Day 1 at 0.5, 1, 2, 4, 6 and 24 hours after the dosing of 100mg/kg of either CBD or CBDA to the animals.

Samples were taken from the jugular vein into lithium heparin. Sampleswere mixed gently by hand then continuously for at least 2 minutes onautomatic mixer and placed in a Kryorack until centrifugation, which wascarried out at approximately 4° C. as soon as practicable. The resultantplasma was separated under low light conditions, transferred to uniquelylabelled clear glass vials, placed in light proof boxes and frozenimmediately at <−50° C.

Toxicokinetic parameters measured included C_(max) (ng/mL), T_(max) (h)and AUC_(0-t) (h*ng/mL) and the results are illustrated in Table 7 forCBDA, Table 8 for CBD (males), Table 9 for CBD (females) and thecomparative C_(max) and AUC_(0-t) are shown in Table 10 for males andTable 11 for females.

Results

TABLE 7 Mean Toxicokinetic Parameters of CBDA are presented below: n = 3Dose of CBDA BDS (mg CBDA/kg/day) 50 100 200 Parameter^(a) Period MalesFemales Males Females Males Females AUC_(0-t) Day 1 55600 149000 80500179000 269000 172000 (h · ng/mL) Day 28 71600 64100 116000 159000 94700156000 Cmax Day 1 19100 21100 24900 38600 35000 27100 (ng/mL) Day 2815700 15000 23400 32500 17700 35900 Tmax Day 1 1 3 1.3 1.7 2.7 1.7 (h)Day 28 1.7 2 1.5 1.7 9 1.3 ^(a)Results are reported as mean unlessstated otherwise

TABLE 8 C_(max) t_(max) t_(1/2) AUC_(0-t) AUC_(0-inf) AUC_(ex) CL/FV_(z)/F Subject (ng/mL) (h) (h) (h * ng/mL) (h * ng/mL) (%) (mL/min/kg)(L/kg) 15 4570 6.0 7.7 51800 60500 14.3 27.5 18.4 16 3620 4.0 5.4 3300035400 6.8 47.1 21.9 17 1400 6.0 8.5 17200 20300 15.1 82.1 60.5 18 24304.0 5.7 28700 31100 7.9 53.6 26.2 19 3090 8.0 n.d. 26400 n.d. n.d. n.d.n.d. 20 3960 6.0 8.6 47300 55300 14.5 30.1 22.3 N 6 6 5 6 5 5 5 5 Mean3180 n.d. 7.2 34100 40500 11.7 48.1 29.9 SD 1140 n.d. 1.5 13100 169004.0 22.0 17.3 Min 1400 4.0 5.4 17200 20300 6.8 27.5 18.4 Median 3360 6.07.7 30800 35400 14.3 47.1 22.3 Max 4570 8.0 8.6 51800 60500 15.1 82.160.5 Geometric 2970 n.d. 7.0 31900 37600 11.1 44.4 27.0 Mean CV % 44.8n.d. 23 42.2 46.9 39.6 46.9 49.5 Geometric Mean

TABLE 9 C_(max) t_(max) t_(1/2) AUC_(0-t) AUC_(0-inf) AUC_(ex) CL/FV_(z)/F Subject (ng/mL) (h) (h) (h * ng/mL) (h * ng/mL) (%) (mL/min/kg)(L/kg) 115 655 2.0 8.4 3000 3280 8.7 508 367 116 2520 2.0 6.6 2000022400 10.6 74.5 42.9 117 1900 8.0 n.d. 22600 n.d. n.d. n.d. n.d. 118 4111.0 4.5 2540 2640 3.7 632 247 119 3270 8.0 n.d. 32400 n.d. n.d. n.d.n.d. 120 3780 6.0 4.7 31300 32800 4.5 50.8 20.8 N 6 6 4 6 4 4 4 4 Mean2090 n.d. 6.1 18600 15300 6.9 316 169 SD 1370 n.d. 1.8 13200 14800 3.3297 167 Min 411 1.0 4.5 2540 2640 3.7 50.8 20.8 Median 2210 4.0 5.721300 12800 6.6 291 145 Max 3780 8.0 8.4 32400 32800 10.6 632 367Geometric 1590 n.d. 5.9 12300 8930 6.3 187 94.8 Mean CV % 113 n.d. 30172 208 54.2 208 238 Geometric Mean

TABLE 10 CBDA (100 mg/kg) CBD (100 mg/kg) Cmax 24,900 3180 AUC_(0-t)80,500 34,100

TABLE 11 CBD level CBDA (100 mg/kg) (100 mg/kg) Cmax 38,600   2090 ng/mLAUC_(0-t) 179,000 18,600 ng/mL * hrConclusions:

It will be apparent from the comparative Tables 10 and 11 that anequivalent amount of CBDA to CBD results in C_(m), and AUC₀₄ valueswhich are very significantly higher (by an order of magnitude) than thatof CBD, suggesting that the CBDA is acting more quickly and is morebioavailable than the CBD. This has significant implications/benefitswhen it comes to treating patients.

OVERALL CONCLUSION

To summarise, the data presented in Examples 1, 2, and 3 demonstratesthat:

CBDA has anticonvulsant effects in a mammalian model of epilepsy and iseffective in treating generalised seizures, more particularly,tonic-clonic seizures. Indeed, this compound appears more effective thanCBD in many of the parameters tested.

CBDA is significantly more potent than CBD upon growth of D. discoideum;and CBDA acts more quickly and is more bioavailable than CBD.

These findings are of great significance as they demonstrate that CBDAoffers an alternative anti-convulsant to CBD. The finding that CBDA ismore potent and more bioavailable than CBD means that a smaller dailydose of the active ingredient may be used in the treatment of epilepsy.In this regard, it appears from Example 3, that doses of less than 400mg and possibly doses of as little as from 1 mg-100 mg, might be used totreat human subjects based on the PK and AUC_(0-t) data of Example 3. Inthis regard, a typical adult patient might weigh 60 kg and thus, a dailydose for such a patient might be from 0.016 mg/kg to 1.6 mg/kg.

REFERENCES

-   Ames F R and Cridland S (1986). “Anticonvulsant effects of    cannabidiol.” S Afr Med J 69:14.-   Chang, P., et al. “The antiepileptic drug valproic acid and other    medium-chain fatty acids acutely reduce phosphoinositide levels    independently of inositol in Dictyostelium.” Dis. Model. Mech. 5.1    (2012): 115-24.-   Chang, P., et al. “Seizure control by ketogenic diet-associated    medium chain fatty acids.” Neuropharmacology 69 (2013): 105-14.-   Chang, P., M. C. Walker, and R. S. Williams. “Seizure-induced    reduction in PIP3 levels contributes to seizure-activity and is    rescued by valproic acid.” Neurobiol. Dis. 62 (2014): 296-306.-   Consroe P, Martin P, Eisenstein D. (1977). “Anticonvulsant drug    antagonism of delta-9-tetrahydrocannabinol induced seizures in    rabbits.” Res Commun Chem Pathol Pharmacol. 16:1-13-   Consroe P, Benedicto M A, Leite J R, Carlini E A, Mechoulam R.    (1982). “Effects of cannabidiol on behavioural seizures caused by    convulsant drugs or current in mice.” Eur J Pharmaco. 83: 293-8-   Cunha J M, Carlini E A, Pereira A E, Ramos O L, Pimental C,    Gagliardi R et al. (1980). “Chronic administration of cannabidiol to    healthy volunteers and epileptic patient.” Pharmacology. 21:175-85-   Cunliffe, Baines, Giachello, Lin, Morgan, Reuber, Russell, Walker    and Williams Epilepsy “Research Methods Update: Understanding the    causes of epileptic seizures and identifying new treatments using    non-mammalian model organisms”. Seizure: European Journal of    Epilepsy. 24C (2015):44-51.-   Eadie, M J (December 2012). “Shortcomings in the current treatment    of epilepsy.” Expert Review of Neurotherapeutics 12 (12): 1419-27.-   Kwan P, Arzimanoglou A, Berg A T, Brodie M J, Hauser W A, Mathern G,    Moshe S L, Perucca E, Wiebe S, French J. (2009) “Definition of drug    resistant epilepsy: Consensus proposal by the ad hoc Task Force of    the ILAE Commission on Therapeutic Strategies.” Epilepsia.-   Mechoulam R and Carlini E A (1978). “Toward drugs derived from    cannabis.” Die naturwissenschaften 65:174-9.-   Porter B E, Jacobson C (December 2013). “Report of a parent survey    of cannabidiol-enriched cannabis use in paediatric treatment    resistant epilepsy” Epilepsy Behaviour. 29(3) 574-7-   Thurman, D J; Beghi, E; Begley, C E; Berg, A T; Buchhalter, J R;    Ding, D; Hesdorffer, D C; Hauser, W A; Kazis, L; Kobau, R; Kroner,    B; Labiner, D; Liow, K; Logroscino, G; Medina, M T; Newton, C R;    Parko, K; Paschal, A; Preux, P M; Sander, J W; Selassie, A;    Theodore, W; Tomson, T; Wiebe, S; ILAE Commission on, Epidemiology    (September 2011). “Standards for epidemiologic studies and    surveillance of epilepsy.” Epilepsia. 52 Suppl 7: 2-26-   Williams, R. S., et al. “Towards a molecular understanding of human    diseases using Dictyostelium discoideum.” Trends Mol. Med. 12.9    (2006): 415-24.

The invention claimed is:
 1. A method of treating epilepsy in a subject,comprising administering to the subject a therapeutically effectiveamount of cannabidiolic acid (CBDA), wherein the CBDA is in the form ofa highly purified extract of cannabis such that the CBDA is present atgreater than 95% of the total extract (w/w) or is syntheticallyproduced.
 2. The method according to claim 1, wherein the epilepsy is ageneralized epilepsy.
 3. The method according to claim 1, wherein theepilepsy is characterized by tonic-clonic seizures.
 4. The methodaccording to claim 1 wherein the therapeutically effective amount is atleast 0.1 mg.
 5. The method according to claim 1, wherein the highlypurified extract comprises less than 1% (w/w) tetrahydrocannabinol (THC)or tetrahydrocannabinolic acid (THCA).
 6. The method according to claim1, wherein the CBDA is administered concomitantly with one or more othercannabinoids.
 7. The method according to claim 6, wherein the one ormore other cannabinoids is cannabidiol (CBD).
 8. The method according toclaim 7, wherein the CBDA:CBD ratio is in the range of from 9:1 to 1:9(CBDA:CBD).
 9. The method according to claim 1, wherein the CBDA isadministered concomitantly with one or more other anti-epileptic drugs(AED).
 10. The method according to claim 1, wherein the CBDA isadministered at a dose of less than 400 mg.
 11. The method according toclaim 10, wherein the CBDA is administered at a dose of from 1 mg to 100mg.
 12. The method according to claim 1, wherein the CBDA is in the formof a highly purified extract of cannabis such that the CBDA is presentat greater than 98% of the total extract (w/w).
 13. The method accordingto claim 7, wherein the CBD is in the form of a highly purified extractof cannabis such that the CBD is present at greater than 95% of thetotal extract (w/w).
 14. The method according to claim 7, wherein theCBD is in the form of a highly purified extract of cannabis such thatthe CBD is present at greater than 98% of the total extract (w/w). 15.The method according to claim 9, wherein the one or more AED is selectedfrom the group consisting of: clobazam; clonazepam, levetiracetam;topiramate; stiripentol; phenobarbital; lacosamide; valproic acid; andzonisamide.
 16. The method of claim 1, wherein said administeringreduces seizure severity.
 17. The method of claim 1, wherein saidadministering reduces the incidence of seizures.
 18. The method of claim17, wherein said administering reduces the incidence of tonic-clonicseizures.
 19. The method of claim 1, wherein said administeringincreases latency to seizure onset.